title: Concerted but noncooperative activation of nucleotide and actuator domains of the Ca-ATPase upon calcium binding.
abstract: Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT(2)). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT(2). Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 A increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K(d) = 4.8 +/- 0.4 microM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.
authors.affiliation: Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
authors.forename: Baowei
authors.initials: B
authors.lastname: Chen
authors.forename: James E
authors.initials: JE
authors.lastname: Mahaney
authors.forename: M Uljana
authors.initials: MU
authors.lastname: Mayer
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Bigelow
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D008958:Models, Molecular; D009068:Movement; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D009711:Nucleotides; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium